The present invention relates to a novel method of purifying plasminogen activator inhibitor and, more particularly, to the use of a modified urokinase for the affinity purification of endothelial cell type plasminogen activator inhibitor.
The serine proteases urinary plasminogen activator (uPA) and tissue plasminogen activator (tPA) convert the zymogen plasminogen into active enzyme plasmin. This enzyme system has been implicated in various physiological and pathological processes including fibrinolysis, inflammation, tissue remodeling, ovulation, trophoblast implantation, tumor invasion and metastasis. A regulatory system exists which controls the conversion of plasminogen to plasmin by inhibiting the plasminogen activators (PAs). Several such inhibitors have been recognized: endothelial cell type PA inhibitor (PAI-1), placental type PA inhibitor (PAI-2), urinary PA inhibitor (PAI-3) and protease nexin.
PAI-1 appears to be the most efficient inhibitor of uPA and tPA among the known PAIs. The second order rate constants have been estimated to be on the order of 10.sup.6 -10.sup.7 M.sup.-1.S.sup.-1. PAI-1 has been found in plasma, platelet, placenta, and a variety of cell cultures. Three forms of PAI-1 have been recognized: (a) an active form; (b) a latent form that can be partially reactivated by treatment with sodium dodecyl sulfate (SDS), guanidinium hydrochloride, urea or phospholipid; and (c) a proteolytically degraded form which can not be reactivated.
PAI-1 was first purified from the conditioned medium of bovine aortic endothelial cell culture by a combination of concanavalin A-Sepharose.RTM. chromatography and SDS-gel electrophoresis, as reported by van Mourik et al., J. Biol. Chem. 259 (23), 14914-14921 (1984). Subsequently, PAI-1 was also isolated from the conditioned media of human fibrosarcoma cell line HT 1080 [Andreasen et al., J. Biol. Chem. 261, 7644-7651 (1986); Nielsen et al., Thromb. Haemostas. 55, 206-212(1986); Kruithof et al., Blood 69(2), 460-466 (1987); Ibid. 70(5), 1645-1653 (1987)], HTC rat hepatoma [Zaheb et al., Thromb. Haemostasis 58, 1017-1023 (1987)], and human endothelial cells [Booth et al., Eur. J. Biochem. 165, 595-600 (1987)]. The isolated PAI-1s consistently show very low specific activity even after denaturant activation [1 .mu.g PAI-1 inhibits 2-5 units (20-50 ng) of urokinase]. This suggests that less than 5% of the molecule was active if it is assumed that the inhibitor forms a 1:1 complex with uPA or tPA. An attempt was made to purify active PAI-1 from a human melanoma cell line, MJZJ, as described by Wagner and Binder, J. Biol. Chem. 261, 14474-14481 (1986). The final product was reported to have a specific activity of 62,000 International Units/mg in the inhibition of tPA which was still ten fold less than expected for fully active PAI-1 (.about.700,000 International Units/mg).
PAI-1 also is known to be produced by the human hepatoma cell line Hep G2 [Sprengers et al., J. Lab. Clin. Med. 105, 751-758 (1985); Wun and Kretzmer, FEBS Lett. 210, 11-16 (1987)].
Further background information on plasminogen activator inhibitors, including PAI-1, can be had by reference to recent reviews on the subject matter such as, for example:
Astedt et al., Fibrinolysis 1, 203-208 (1987); Sprengers and Kluft, Blood 69, 381-387 (1987); and Kruithof, Fibrinolysis 2, 59-70 (1988).